RESUMO
Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O(2) levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O(2) imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O(2) in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O(2) levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O(2) in 3D tissue models.
Assuntos
Medições Luminescentes/métodos , Imagem Molecular/métodos , Sondas Moleculares/metabolismo , Nanopartículas/metabolismo , Neurônios/química , Oxigênio/análise , Fatores Etários , Animais , Sondas Moleculares/química , Estrutura Molecular , Nanopartículas/química , RatosRESUMO
We compared three optical platforms for measurement of cellular respiration: absolute oxygen consumption rates (OCRs) in hermetically sealed microcuvettes, relative OCRs measured in a 96-well plate with oil seal, and steady-state oxygenation of cells in an open 96-well plate. Using mouse embryonic fibroblasts cell line, the phosphorescent intracellular O2 probe MitoXpress-Intra, and time-resolved fluorescence reader, we determined algorithms for conversion of relative OCRs and cell oxygenation into absolute OCRs, thereby allowing simple high-throughput measurement of absolute OCR values.
Assuntos
Respiração Celular , Dispositivos Ópticos , Consumo de Oxigênio , Algoritmos , Animais , Calibragem , Células Cultivadas , Corantes Fluorescentes , CamundongosRESUMO
Measurement of cell oxygenation and oxygen consumption is useful for studies of cell bioenergetics, metabolism, mitochondrial function, drug toxicity and common pathophysiological conditions. Here we present a new platform for such applications which uses commercial multichannel biochips (µ-slides, Ibidi) and phosphorescent O2 sensitive probes. This platform was evaluated with both extracellular and intracellular O2 probes, several different cell types and treatments including mitochondrial uncoupling and inhibition, depletion of extracellular Ca(2+) and inhibition of V-ATPase and histone deacetylases. The results show that compared to the standard microwell plates currently used, the µ-slide platform provides facile O2 measurements with both suspension and adherent cells, higher sensitivity and reproducibility, and faster measurement time. It also allows re-perfusion and multiple treatments of cells and multi-parametric analyses in conjunction with other probes. Optical measurements are conducted on standard fluorescence readers and microscopes.
Assuntos
Substâncias Luminescentes/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Oxigênio/metabolismo , Animais , Respiração Celular , Humanos , Camundongos , Técnicas Analíticas Microfluídicas/normas , Ratos , Padrões de ReferênciaRESUMO
Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.
Assuntos
Bioimpressão , Esferoides Celulares , Microscopia , Oxigênio/metabolismo , Reprodutibilidade dos Testes , Esferoides Celulares/metabolismoRESUMO
Monitoring of oxygenation is important for physiological experiments investigating the growth, differentiation and function of individual cells in 3D tissue models. Phosphorescence based O2 sensing and imaging potentially allow this task; however, current probes do not provide the desired bio-distribution and analytical performance. We present several new cell-penetrating phosphorescent conjugates of a Pt(ii)-tetrakis(pentafluorophenyl)porphine (PtPFPP) dye produced by click-modification with thiols, and perform their evaluation as O2 imaging probes for 3D tissue models. The hydrophilic glucose (Pt-Glc) and galactose (Pt-Gal) conjugates demonstrated minimal aggregation and self-quenching in aqueous media, and efficient in-depth staining of different cell types and multi-cellular aggregates at working concentrations ≤10 µM. The Pt-Glc probe was applied in high-resolution phosphorescence lifetime based O2 imaging (PLIM) in multi-cellular spheroids of cancer cells (PC12), primary neural cells (neurospheres) and slices of brain tissue, where it showed good analytical performance, minimal effects on cell viability and appropriate responses to O2 with phosphorescence lifetimes changing from 20 µs in air-saturated to 57 µs under deoxygenated conditions. In contrast, mono- and tetra-substituted oligoarginine conjugates of PtPFPP showed marked aggregation and unstable photophysical properties precluding their use as O2 sensing probes.